Animal cell culture, notably mammalian cell culture, is preferably used for the expression of recombinantly produced, glycosylated proteins for therapeutic and/or prophylactic applications. Glycosylation patterns of recombinant glycoproteins are important, because the oligosaccharide side chains of glycoproteins affect protein function, as well as the intramolecular interactions between different regions of a protein. Such intramolecular interactions are involved in protein conformation and tertiary structure of the glycoprotein. (See, e.g., A. Wittwer et al., 1990, Biochemistry, 29:4175-4180; Hart, 1992, Curr. Op. Cell Biol., 4:1017-1023; Goochee et al., 1991, Bio/Technol., 9:1347-1355; and R. B. Parekh, 1991, Curr. Op. Struct. Biol., 1:750-754). In addition, oligosaccharides may function to target a particular polypeptide to certain structures based on specific cellular carbohydrate receptors. (M. P. Bevilacqua et al., 1993, J. Clin. Invest., 91:379-387; R. M. Nelson et al., 1993, J. Clin. Invest, 91:1157-1166; K. E. Norgard et al., 1993, Proc. Natl. Acad. Sci. USA, 90:1068-1072; and Y. Imai et al., 1993, Nature, 361-555-557).
The terminal sialic acid component of a glycoprotein oligosaccharide side chain is known to have an effect on numerous aspects and properties of a glycoprotein, including absorption, solubility, thermal stability, serum half life, clearance from the serum, as well as its physical and chemical structure/behavior and its immunogenicity. (A. Varki, 1993, Glycobiology, 3:97-100; R. B. Parekh, Id., Goochee et al., Id., J. Paulson et al., 1989, TIBS, 14:272-276; and A. Kobata, 1992, Eur. J. Biochem., 209:483-501; E. Q. Lawson et al., 1983, Arch. Biochem. Biophys., 220:572-575; and E. Tsuda et al., 1990, Eur. J. Biochem., 188:405-411).
In general, protein expression levels in mammalian cell culture-based systems are considerably lower than in microbial expression systems, for example, bacterial or yeast expression systems. However, bacterial and yeast cells are limited in their ability to optimally express high molecular weight protein products, to properly fold a protein having a complex steric structure, and/or to provide the necessary post-translational modifications to mature an expressed glycoprotein, thereby affecting the immunogenicity and clearance rate of the product.
As a consequence of the limitations of the culturing of animal or mammalian cells, particularly animal or mammalian cells which produce recombinant products, the manipulation of a variety of parameters has been investigated, including the employment of large-scale culture vessels; altering basic culture conditions, such as incubation temperature, dissolved oxygen concentration, pH, and the like; the use of different types of media and additives to the media; and increasing the density of the cultured cells. In addition, process development for mammalian cell culture would benefit from advances in the ability to extend run times to increase final product concentration while maintaining high product quality. An important product quality parameter is the degree and completeness of the glycosylation structure of a polypeptide product, with sialic acid content commonly used as a measure of glycoprotein quality.
Run times of cell culture processes, particularly non-continuous processes, are usually limited by the remaining viability of the cells, which typically declines over the course of the run. The maximum possible extension of high cell viabilities is therefore desired. Product quality concerns also offer a motivation for minimizing decreases in viable cell density and maintaining high cell viability, as cell death can release sialidases to the culture supernatant, which may reduce the sialic acid content of the protein expressed. Protein purification concerns offer yet another motivation for minimizing decreases in viable cell density and maintaining high cell viability. The presence of cell debris and the contents of dead cells in the culture can negatively impact on the ability to isolate and/or purify the protein product at the end of the culturing run. By keeping cells viable for a longer period of time in culture, there is thus a concomitant reduction in the contamination of the culture medium by cellular proteins and enzymes, e.g., cellular proteases and sialidases, that can cause degradation and ultimate reduction in quality of the desired glycoprotein produced by the cells.
Various parameters have been investigated to achieve high cell viability in cell cultures. One parameter involved a single lowering of the culture temperature following initial culturing at 37° C. (for example, Roessler et al., 1996, Enzyme and Microbial Technology, 18:423-427; U.S. Pat. Nos. 5,705,364 and 5,721,121 to T. Etcheverry et al., 1998; U.S. Pat. No. 5,976,833 to K. Furukawa et al., 1999; U.S. Pat. No. 5,851,800 to L. Adamson et al.; WO 99/61650 and WO 00/65070 to Genentech, Inc.; WO 00/36092 to Biogen, Inc.; and U.S. Pat. No. 4,357,422 to Girard et al.).
“Other parameters investigated involved the addition of components to the culture. The growth factor inhibitor suramin was shown to prevent apoptosis during exponential growth of CHO (Chinese Hamster Ovary) K1:CycE cells (Zhangi et al., Biotechnol. Prog. 2000, 16, 319-325). However, suramin did not protect against apoptosis during the death phase. As a result, suramin was capable of maintaining high viability during the growth phase, but did not allow for an extension of culture longevity. The same authors report that for the CHO 111-10PF cell line, dextran sulfate and polyvinyl sulfate could, similarly to suramin, increase day 3 viable cell density and viability relative to the control culture. The effect of dextran sulfate or polyvinyl sulfate during the death phase was however not reported. Suramin, dextran sulfate and polyvinyl sulfate were also reported to be effective at preventing cell aggregation.”
Heparin has been supplemented to animal cell culture media in order to adapt anchorage-dependant cell lines to suspension conditions (e.g. U.S. Pat. No. 5,348,877 to McKenna and Granados, 1994). Heparin is also known to bind to growth factors, such as the heparin-binding EGF-like growth factor (HB-EGF; Raab and Klagsbrun, Biochim. Biophys. Acta 1997, 1333, F179-F199). Cell surface heparan sulfate proteoglycans (HSPG) reportedly enhance HB-EGF binding and bioactivity for certain cell types including wild-type CHO cells (Raab and Klagsbrun, 1997). [Heparan sulfate only differs from heparin in that it has fewer N- and O-sulfate groups and more N-acetyl groups (McKenna and Granados, 1994). For the purpose of this disclosure, heparin and heparan sulfate are considered equivalent and will generically be referred to as heparin.] It has been proposed, for the heparin-binding growth factor FGF-2, that binding to HSPG increases the local FGF-2 concentration on the cell surface, which in turn increases the probability of FGF-2 binding to the tyrosine kinase receptors of the cells (Raab and Klagsbrun, 1997). It has been shown that pentosan polysulfate can block the action of heparin-binding growth factors on cultured cells (Zugmaier et al., J. Nat. Cancer Inst. 1992, 84, 1716-1724.
Patent literature on the use of dextran sulfate in animal cell culture pertain to the supplementation of dextran sulfate to a medium in order: 1) To improve growth rate and increase the number of population doublings before senescence for human endothelial cells (U.S. Pat. Nos. 4,994,387 and 5,132,223 to Levine et al., 1991, 1992); 2) To increase recombinant protein yield in mammalian cell lines (U.S. Pat. No. 5,318,898 to Israel, 1994); 3) To induce single cell suspension in insect cell lines (U.S. Pat. No. 5,728,580 to Shuler and Dee, 1996); 4) To increase growth-promoting activity of human hepatocyte-growth factor and to suppress its degradation (U.S. Pat. Nos. 5,545,722 and 5,736,506 to Naka, 1996 and 1998); 5) To increase viable cell density and recombinant protein expression (WO 98/08934 to Gorfien et al., 1997).
In all reported cases referring to the presence or supplementation of dextran sulfate in a medium, dextran sulfate was present throughout the culture time in that given medium. In no case were the benefits of a delayed addition reported. Moreover, it has never been reported that dextran sulfate can delay the onset of the death phase, extend the growth phase, or arrest the death phase.
With increasing product concentration in the culture, it can be observed in cell culture processes that the product quality decreases, as determined by the measured sialic acid content of the oligosaccharide glycostructure. Usually, a lower limit for an acceptable sialic acid content exists as determined by drug clearance studies. High abundance of a protein produced by cells in culture is optimally accompanied by high quality of the protein that is ultimately recovered for an intended use. Such high protein quality is correlated with the degree and completeness of a protein's glycosylation structure, with sialic acid content used as a measure of these parameters.
It has been reported that the addition of D-galactose (4 g/L) to the basal medium of a small scale cell culture, e.g., 2 liter (2L) bioreactors, increased galactosylation of the heavy chain of a monoclonal antibody (MAb) produced by the cultured cells. (S. Weikert et al., “Engineering CHO Cells to Maximize Sialic Acid Content of Recombinant Protein”, presentation at the “Protein Expression” Meeting, sponsored by the Cambridge Healthtech Institute, Apr. 5-6, 2001, McLean, Va.). As reported, galactose was provided as a one-time additive to small scale cultures, and not as a feeding medium additive provided throughout the entire culture period. The report contains no recognition of the obstacles involved in the sialylation of a glycoprotein produced in cultures maintained on a large scale (e.g., greater than 2 L).
WO 01/59075 A1 (Genentech, Inc.) discloses a process for improved or enhanced sialylation of glycoproteins by introducing into cells in culture a nucleic acid sequence encoding a mutated UDP-GlcNac2-epimerase, which is a rate-limiting enzyme in the biosynthetic pathway of a specific nucleotide sugar, CMP sialic acid. In a similar manner, X. Gu and D. Wang (1998, Biotech. Bioeng., 58(6):642-648) describe supplementing culture medium with ManNac, a specific precursor for sialic acid synthesis, to increase the availability of CMP-sialic acid and improve product sialylation.
Similar to WO 01/59075 A1, X. Gu and D. I. C. Wang (1998, Biotech. Bioeng., 58(6):642-648) disclose that the supplementation of culture medium with N-acetylmannosamine (ManNAc) increased the CMP-sialic acid pool in the cells and ultimately the sialic acid content of the recombinantly produced human IFN-γ glycoprotein in cultures of 20 mL scale. ManNAc supplementation involved an initial addition of this component to the cultures prior to sialylation analysis of the produced product after 96 hours of culture.
WO 01/59089 A2 (Genentech, Inc.) discloses enhancement of intracellular CAD activity in glycoprotein-producing cells made resistant to CAD inhibitors. This published international application discloses that CAD refers to the multienzyme polypeptide complex (carbamoyl phosphate synthetase (CPS II), aspartate transcarbamoylase and dihydro-orotase) that catalyzes the first three reactions in the de novo biosynthesis of pyrimidines, particularly UMP, which is converted to UTP. CAD-resistant cells are reported to have increased UTP pools, and increased UDP-galactose levels along with increased glycosylation of the protein products; UDP-galactose is a known substrate in the glycosylation pathway.
Recombinantly produced protein products that are properly glycosylated and sialylated are increasingly becoming medically and clinically important for use as therapeutics, treatments and prophylactics. Therefore, the development of reliable cell culture processes that economically and efficiently achieve an increased final protein product concentration, in conjunction with a high level of product quality, such as is determined by sialic acid content, fulfills both a desired and needed goal in the art.